Since glypican 3 is highly expressed in liver cancer with frequency, the expression profile analysis of glypican 3 in liver cancer is thought to be probably useful for the functional identification of glypican 3 in liver cancer, the treatment or diagnosis of liver cancer, and the prognostic prediction of liver cancer. For the general expression analysis of proteins, immunohistochemistry, particularly, enzyme antibody technique, is widely used in pathological diagnosis. The immunohistochemistry is a method for detecting, highly sensitively and specifically, the presence and distribution of a substance (antigen) in vivo using biological reagents such as antibodies or enzymes. Examples of the features of the immunohistochemistry include: 1. its procedures are convenient; 2. the method is widely applicable in such a way that the obtained biochemical information can be applied to morphological information; 3. the method provides biologically and pathologically important information; and 4. care different from usual methods should be taken because the method uses biological reagents. Moreover, immunohistochemical staining also has the advantage that it can be used in the pathological diagnosis or morphological observation of interest using a wide range of samples such as fresh frozen sections, cytological samples, and paraffin sections of fixed tissues.
Among immunohistochemical methods, immunostaining based on the enzyme antibody technique can utilize formalin-fixed paraffin-embedded tissues used in usual pathological diagnosis and is therefore applied to a very wide range. However, unless sufficient care is taken to, for example, possible artifacts derived from formalin-fixed tissues themselves, not only does staining end unsuccessfully, but also such artifacts sometimes bring about alteration of antigens attributed to formalin fixation and embedding or alteration of antibody penetration or reactivity, resulting in false positive or false negative. Such false positive or false negative may end in misinterpretation of staining results.
Formalin fixation used in usual histological search is useful for maintaining morphology but is not an ideal fixative from the viewpoint of maintaining affinity for antibodies. Thus, alcohol or the like is also used instead of formalin as a fixative. However, a fixation method has not yet been established which is excellent in maintaining morphology and can preserve the affinity of every antigen for antibodies. Accordingly, the formalin fixation is a practical fixation method widely used, although it has a problem in maintaining affinity for antibodies.
Thus, some methods have been proposed for weakening the influence of formalin fixation on the maintenance of affinity for antibodies. A first possible strategy is to select an antibody recognizing an epitope unsusceptible to formalin fixation and use the antibody in immunostaining. Such an antibody recognizing an antigen epitope unsusceptible to formalin fixation, selected from among antibodies recognizing the same epitope can be used in immunostaining to thereby reduce false negative. However, glypican 3 undergoes post-translational modification by protease or the like after being expressed on cell surface and therefore has limitations on epitopes capable of binding to antibodies. Thus, this approach lacks epitope diversity for selecting a suitable epitope.
A second method is to enhance the sensitivity of immunostaining to thereby detect an antigen that cannot be visualized by a usual method. The simplest method is a method which involves adding heavy metal such as copper to DAB (3,3′-diaminobenzidine tetrahydrochloride) usually used for color development in immunostaining. However, this method cannot be expected to significantly increase sensitivity. A method, such as ABC (avidin-biotinylated peroxidase complex) method or LSAB (labeled streptavidin biotinylated antibody) method, has been attempted which involves enhancing sensitivity by repetitively reacting a biotinylated secondary antibody and ABC or enzyme-labeled avidin with sections. However, it has been confirmed that as the number of reactions increases, non-specific background staining also tends to increase. Furthermore, EPOS (enhanced polymer one-step staining) method using an enzyme-labeled dextran polymer or CSA (catalyzed signal amplification) method combining biotinylated tyramide with ABC method has become available, dramatically improving the sensitivity of staining. However, in the detection of antigens in formalin-fixed tissues, the use of the conventional method such as ABC method allows diagnosis of tumor tissues as being positive and normal or non-tumor tissues as being negative, on the basis of which tumor tissues can be differentiated from non-tumor tissues, whereas the use of the highly sensitive method provides detection of even a trace amount of antigens in non-tumor tissues and may therefore fail to make such differentiation depending on the types of antigens. Moreover, the highly sensitive method used also had the problem of increased background staining, because of having high sensitivity.
A third method is to retrieve the reactivity of an antigen whose reactivity with antibodies has been reduced due to formalin fixation. In a method introduced in the 1970s, which involves digesting sections with protease (protease-induced epitope retrieval method; hereinafter, referred to as “PIER method” or “protease-induced epitope retrieval method”), sections are digested with trypsin, pepsin, or the like, prior to immunostaining. This method had such problems as peel-off of sections from glass attributed to the digestion of the sections themselves, and unstable staining results.
Thereafter, heat-induced epitope retrieval method (hereinafter, referred to as “HIER method” or “heat-induced epitope retrieval method”) was developed in the 1990s. Heating using a microwave, boiling, or an autoclave allegedly enables an epitope to bind to antibodies as a result of hydrolyzing the antigen by the high-temperature treatment. The expression of glypican 3 in liver cancer has also been detected so far by the HIER method (Non-Patent Documents 1 to 5 and Patent Document 1). However, since anti-glypican 3 antibodies exhibit cross-reactivity with the epithelial cells of blood vessels or hepatic sinusoids, this approach requires such complicated treatment that blocking reaction with a normal liver cell-derived protein lysate is performed in advance to exclude such cross-linking (Non-Patent Document 4). Thus, the HIER method conventionally used cannot accurately detect glypican 3 in liver cancer tissues in such a way that glypican 3 originally expressed on the cell membrane is observed as if this antigen is cytoplasmically expressed (Non-Patent Document 2). Accordingly, an accurate detection method for the expression has been demanded to be developed as a substitute for the conventional HIER method.    [Non-Patent Document 1] Capurro M, Wanless I R, Sherman M, Deboer G, Shi W, Miyoshi E, Filmus J., (2003) Gastroenterology 125 (1), 89-97    [Non-Patent Document 2] Yamauchi N, Watanabe A, Hishinuma M, Ohashi K, Midorikawa Y, Morishita Y, Niki T, Shibahara J, Mori M, Makuuchi M, Hippo Y, Kodama T, Iwanari H, Aburatani H, Fukayama M., (2005) Mod Pathol 18 (12), 1591-8    [Non-Patent Document 3] Libbrecht L, Severi T, Cassiman D, Vander Borght S, Pirenne J, Nevens F, Verslype C, van Pelt J, Roskams T., (2006) Am J Surg Pathol 30 (11), 1405-11    [Non-Patent Document 4] Grozdanov P N, Yovchev M I, Dabeva M D., (2006) Lab Invest 86 (12), 1272-84    [Non-Patent Document 5] Llovet, J. M., Chen, Y., Wurmbach, E., Roayaie, S., Fiel, M. I., Schwartz, M., Thung, S. N., Khitrov, G., Zhang, W., Villanueva, A., Battiston, C., Mazzaferro, V., Bruix, J., Waxman, S., Friedman, S. L., (2006) Gastroenterology 131 (6), 1758-1767    [Patent Document 1] WO2003100429    [Patent Document 2] WO2006006693    [Patent Document 3] WO2004022739